EP2460873A1

EP2460873A1 - A method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family with attenuated expression of any of the cynT, cynS, cynX or cynR genes or a combination thereof

Info

Publication number: EP2460873A1
Application number: EP20120157893
Authority: EP
Inventors: Dmitriy Vladimirovich Filippov; Elvira Borisovna Voroshilova; Mikhail Markovich Gusyatiner
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 2006-12-12
Filing date: 2007-12-03
Publication date: 2012-06-06
Anticipated expiration: 2027-12-03
Also published as: RU2006143864A; US20090269819A1; EP2089528A1; US7919283B2; EP2460873B1; WO2008072640A1; ATE556146T1; EP2089528B1

Abstract

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to attenuate expression of one or more genes selected from the group consisting of cynT, cynS, cynX and cynR.

Description

Technical Field

The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of the cynTSX operon and the cynR gene.

Background Art

Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, US patent No. 4,278,765 ). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042 , or US patent Nos. 4,346,170 ; 5,661,012 and 6,040,160 ).
Another way to enhance L-amino acid production yields is to attenuate expression of a gene, or several genes involved in degradation of the target L-amino acid, genes diverting the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of carbon, nitrogen, and phosphate fluxes, and genes coding for toxins etc.
It is known that cyanate, a toxic compound, is the product of the spontaneous dissociation of urea in solution as well as of the decomposition of carbamoyl phosphate, an intermediate in the pyrimidine and arginine biosynthetic pathways. In Escherichia coli, the process of cyanate detoxication is mediated throught the cynTSX operon that enables E. coli to degrade and use cyanate as a sole nitrogen source. (SungY.-C. et.al., J. Biol. Chem.;263(29):14769-14775 (1988)). The cynTSX operon which contains three genes: cynT, cynS, and cynX is transcribed as a policistronic message. The cynTSX operon is inducible by cyanate and is under the control of the inducer gene cynR. This operon is also subject to catabolite repression by bicarbonate.
The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn²⁺/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate (Guilloton M.B. et.al., J. Biol. Chem.;267(6):3731-4 (1992)).
Cyanase CynS is composed of 8-10 identical subunits (Mr = 17,008) with significant amount of α-helix and β-sheet structure which catalytic activity links to the structural integrity of the oligomer (Little R.M. et a., J. Biol. Chem.;262(21):10120-10126 (1987)).
The cynX gene encodes hydrophobic protein CynX with undetermined function. Based exclusively on degrees of sequence similarity, CynX is determined as a member of the major facilitator superfamily (MFS), which is one of the two largest families of membrane transporters and is present ubiquitously in bacteria, archaea, and eukarya, as putative cyanate transporter (Pao S.S., Microbiol. And Mol. Biol. Rev.;62(1):1-34 (1998)).
The physiological role of cynT, cynS and cynX was investigated by construction of chromosomal mutants in which these genes were rendered inactive. The delta cynT chromosomal mutant expressed an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain was inhibited by cyanate, and the mutant strain was unable to degrade cyanate and therefore could not use cyanate as the sole nitrogen source when grown at a partial CO₂ pressures (pCO₂) of 0.03% (air). At a high pCO₂ (3%), however, the delta cynT strain behaved like the wild-type strain; it was significantly less sensitive to the toxic effects of cyanate and could degrade cyanate and use cyanate as the sole nitrogen source for growth. These results are consistent with the proposed function for carbonic anhydrase (Guilloton M.B. et.al., J. Bacteriol.;175(5):1443-51(1993)).
It was found that a carbonic anhydrase, YadF, is essential for cell growth in the absence of another carbonic anhydrase, CynT, in Escherichia coli. However, mutant strains lacking both of them grew at high CO₂ concentrations (5%), where non-enzymatic mechanisms generate HCO₃ ^-. This suggests that these carbonic anhydrases are essential because they maintain HCO₃ ^- levels at ambient CO₂ concentrations (Hashimoto M., and Kato J., Biosci. Biotechnol. Biochem.;67(4):919-22 (2003)).
CynT is the single E. coli Can (previously YadF) paralog, it can, when induced with azide, replace Can (Merlin C.,et.al., J. Bacteriol.;185(21):6415-24 (2003)).
Expression of the cynTSX operon is controlled by the CynR protein encoded by the cynR gene and transcribed in the direction opposite to that of the cyn operon (Lamblin A.-F.J., et.al., J. Bacteriol.;175(21):7990-7999 (1993)). Analysis of β-galactosidase transcriptional fusions suggest that CynR is a dual transcriptional regulator that belongs to the LysR family and is negatively autoregulated independently of cyanate (Sung Y.-C., et al., J. Bacteriol.; 174(11):3645-3650 (1992)). CynR is 32,000 Mr protein that recognizes a 136-bp DNA fragment encompassing the cynR-cynTSX intergenic region. Circular permutation assays indicated that CynR induces bending of the DNA upon binding and that this bending decreases in the presence of cyanate (Lamblin A.-F.J., et.al., J. Bacteriol.;176(21):6613-6622 (1994)).
But currently, there have been no reports of attenuating expression of the cynTSX operon and the cynR gene for the purpose of producing L-amino acids.

Summary of the Invention

Objects of the present invention include enhancing the productivity of L-amino acid-producing strains and providing a method for producing an L-amino acid using these strains.
The above objects were achieved by finding that attenuating expression of the cynTSX operon and the cynR gene can enhance production of L-amino acids, such as L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.
The present invention provides a bacterium of the Enterobacteriaceae family which has an increased ability to produce amino acids, such asL-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-phenylalanine. L-tyrosine, and L-tryptophan.
It is an object of the present invention to provide an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of one or more of the genes selected from the group consisting of cynT, cynS, cynX and cynR.
It is a further object of the present invention to provide the bacterium as described above, wherein the expression of one or more genes selected from the group consisting of cynT, cynS, cynX and cynR is attenuated by inactivation of the one or more genes.
It is a further object of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Escherichia.
It is a further object of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Pantoea.
It is a further object of the present invention to provide the bacterium as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
It is a further object of the present invention to provide the bacterium as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
It is a further object of the present invention to provide the bacterium as described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.
It is a further object of the present invention to provide a method for producing an L-amino acid comprising: - cultivating the bacterium as described above in a medium, and - collecting said L-amino acid from the medium.
It is a further object of the present invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
It is a further object of the present invention to provide the method as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
It is a further object of the present invention to provide the method as described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.
The present invention is described in detail below.

Detailed Description of the Preferred Embodiments

1. Bacterium of the present invention

The bacterium of the present invention is an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of one or more of the genes selected from the group consisting of cynT, cynS, cynX and CynR.
In the present invention, "L-amino acid-producing bacterium" means a bacterium which has an ability to produce and secrete an L-amino acid into a medium, when the bacterium is cultured in the medium.
The term "L-amino acid-producing bacterium" as used herein also means a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain of the bacterium, for example, E. coli, such as E. coli K-12, and preferably means that the bacterium is able to cause accumulation in a medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L, of the target L-amino acid. The term "L-amino acid" includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
The term "aromatic L-amino acid" includes L-phenylalanine, L-tyrosine, and L-tryptophan. The term "non-aromatic L-amino acid" includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline, and L-arginine are particularly preferred.
The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc.. Specifically, those classified into the Enterobacteriaceae family according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. A bacterium belonging to the genus Escherichia or Pantoea is preferred.
The phrase "a bacterium belonging to the genus Escherichia" means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. coli).
The bacterium belonging to the genus Escherichia that can be used in the present invention is not particularly limited, however for example, bacteria described by Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) are encompassed by the present invention.
The phrase "a bacterium belonging to the genus Pantoea" means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on the nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).
The phrase "bacterium has been modified to attenuate expression of a gene(s)" means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount ofprotein(s) encoded by the gene(s) as compared with an unmodified bacterium, or the modified bacterium is unable to synthesize protein(s) encoded by the gene(s). The phrase "bacterium has been modified to attenuate expression of a gene(s)" also include that the bacterium has been modified in such a way that the modified gene encodes a mutant protein(s) which has/have a decreased activity.
The phrase "inactivation of gene(s)" means that the modified gene(s) encode(s) completely inactive protein(s). It is also possible that the modified DNA region is unable to naturally express the gene due to a deletion of a part of the gene or the gene entirely, shifting of the reading frame of the gene, introduction of missense/nonsense mutation(s), or modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc.
The cynTSX operon is transcribed as a policistronic mRNA including messages from all three genes. So modification of first cynT gene, such as deletion of the whole or a part of the cynT gene, introducing stop codon or frame shift, could also lead to inactivation of downstream genes.
The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various known methods including Northern blotting, quantitative RT-PCR, and the like.
The cynTSX operon includes three genes in the following order. The cynT gene (synonyms - ECK0336, b0339) encodes the CynT protein, carbonic anhydrase monomer (synonym - B0339). The cynT gene (nucleotide positions: 358,023 to 358,682; GenBank accession no. NC_000913.2; gi: 49175990) is located between the CynR gene and the cynS gene on the E. coli K-12 chromosome. The cynS gene (synonyms - ECK0337 , b0340 , cnt , cyaN) encodes the CynS protein, cyanase monomer (synonyms - B0340, Cnt, CyaN, CynS, cyanate hydrolase, cyanase, cyanate C-N-lyase). The cynS gene (nucleotide positions: 358,713 to 359,183; GenBank accession no. NC_000913.2; gi: 49175990) is located between the cynT gene and the cynX gene on the E. coli K-12 chromosome. The cynX gene (synonyms - ECK0338 , b0341) encodes the CynX protein, putative cyanate MFS transporter (synonym - B0341) . The cynX gene (nucleotide positions: 359,216 to 360,370; GenBank accession no. NC_000913.2; gi: 49175990) is located between the cynS gene and the lacA gene on the E. coli K-12 chromosome. Examples of the genes of cynT, cynS, and cynX from Escherichia coli are represented by SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively. The amino acid sequences encoded by the genes of cynT, cynS, and cynX are presented by SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectivly.
The cynR gene (synonyms - ECK0335 , b0338) encodes the CynR protein, dual transcriptional regulator that belongs to the LysR family (synonyms - B0338, CynR, cyn operon positive regulator). The cynR gene (nucleotides complementary to nucleotides 357015 to 357914 in the sequence of GenBank accession no. NC_000913.2; gi: 49175990) is located between the codA gene and the cynT gene on the E. coli K-12 chromosome. The nucleotide sequence of the cynR gene and the encoded CynR amino acid sequence are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
Since there may be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the gene to be inactivated on the chromosome is not limited to the genes shown in SEQ ID Nos:1, 3, 5 and 7 but may include genes homologous to SEQ ID Nos:1, 3, 5 and 7 which encode variant proteins of the CynT, CynS, CynX and CynR proteins. The phrase "variant proteins" as used in the present invention means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids.
The number of changes in the variant proteins depends on the position in the three dimensional structure of the protein or the type of amino acid residues. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQ ID NOs: 2, 4, 6 and 8. These changes in the variants are conservative mutations that preserve the function of the protein. In other words, these changes in the variants can occur in regions of the protein which are not critical for the three dimensional structure of the protein. This is because some amino acids have high homology to one another so the three dimensional structure is not affected by such a change. A conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp, Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile. Val, if the substitution site is a hydrophobic amino acid; between Gln, Asn, if it is a polar amino acid; among Lys, Arg, His, if it is a basic amino acid; between Asp, Glu, if it is an acidic amino acid; and between Ser, Thr, if it is an amino acid having a hydroxyl group. Typical conservative mutations are conservative substitutions. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln, substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, Ile or Leu for Val. Substitutions, deletions, insertions, additions, or inversions and the like of the amino acids described above include naturally occurred mutations (mutant or variant) depending on differences in species, or individual differences of microorganisms that retain the cynT, cynS, cynX or cynR gene. Such a gene can be obtained by modifying the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, or 7 using, for example, site-directed mutagenesis, so that the site-specific amino acid residue in the protein encoded includes substitutions, deletions, insertions, or additions.
Moreover, the protein variant encoded by the cynT, cynS, cynX and cynR may have a homology of not less than 80 %, preferably not less than 90%, and most preferably not less than 95 %, with respect to the entire amino acid sequences shown in SEQ ID NO. 2, 4, 6 and 8 respectively as long as the activity of the CynT, CynS, CynX and CynR proteins prior to inactivation is maintained.
Activity of carbonic anhydrase, cyanase and cyanate MFS transporter can be measured by the method described in (Guilloton M.B. et,al., J. Biol. Chem.;267(6):3731-4 (1992), Guilloton M.B. et.al., J.Bacteriol.;175(5):1443-51(1993)).
Positive regulation of the cynTSX operon and the negative autoregulation of the cynR gene by the dual transcriptional regulator CynR can be assessed by the method described in (Sung Y.-C. et al., J.Bacteriol.; 174(11):3645-3650 (1992)).
Moreover, either of the cynT, cynS, cynX and cynR gene may be a variant which hybridizes with the nucleotide sequence shown in SEQ ID NO: 1, 3, 5 and 7 respectively or a probe which can be prepared from the nucleotide sequences under stringent conditions. "stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, still more preferably not less than 90%, and most preferably not less than 95%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1 X SSC, 0.1% SDS, preferably 0.1 X SSC, 0.1% SDS at 60°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times. The length of the probe may be suitably selected, depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0.
Expression of the cynT, cynS, cynX and cynR genes can be attenuated by introducing a mutation into these genes on the chromosome so that the intracellular amount of the CynT, CynS, CynX and CynR proteins encoded by these genes is decreased as compared to an unmodified strain. Such a mutation can be introduction of insertion of a drug-resistance gene, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M.F., J. Biol. Chem., 272, 8611-8617 (1997); Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). Expression of the cynT, cynS, cynX and cynR genes can also be attenuated by modifying expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. ( WO95/34672 Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).
For example, the following methods may be employed to introduce a mutation by gene recombination. A mutant gene encoding a mutant protein having a decreased activity is prepared, and the bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected. Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B.L., Proc. Nat1. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication ( U.S. Patent Nos. 6,303,383 and 5,616,480 ). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid lacking the ability to replicate in the host.
Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene ( U.S. Patent No. 5,175,107 ), or by conventional methods, such as mutagenesis treatment with UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).
The delta cynT chromosomal mutant expresses an active cyanase but no active carbonic anhydrase. In contrast to the wild-type strain, the growth of the delta cynT strain is inhibited by cyanate, and the mutant strain is unable to degrade cyanate and therefore can not use cyanate as the sole nitrogen source when grown at a partial CO₂ pressures (pCO₂) of 0.03% (air).
The delta cynS chromosomal mutant expresses an active carbonic anhydrase but no active cyanase. The delta cynS strain has sensitivity to cyanase activity to a lesser extent than delta cynT chromosomal mutant. A major physiological effect of cyanate on growth is inhibition of carbamoyl phosphate synthetase, which catalyze first step in both arginine and pyrimidine biosynthesis. So the addition of arginine plus uracil significantly relieves inhibition by cyanate for both the delta cynS chromosomal mutant and wild-type strains but not for the delta cynT chromosomal mutant because depletion of bicarbonate in the delta cynT chromosomal mutant strain with induced cyanase activity at low pCO₂ is more serious than the direct inhibitory effect of cyanate on growth.
No change in the phenotype compared with the parental strain was observed in the delta cynX chromosomal mutant except for slightly reduced levels of expression of cyanase by the mutant strains that may be related to effects on the rates of transcription or on the stability of the transcript.
The chromosomal mutant with double mutation delta cynTSX and delta galk and plasmids containing an insert with the cynR-galK transcriptional fusion sequence expresses GalK activity when cyanate is present.
Therefore, the reduced or absent activity of the CynT, CynS, CynX and CynR proteins in the bacterium according to the present invention can be determined when compared to the parent unmodified bacterium. The presence or absence of the cynT, cynS, cynX and cynR genes in the chromosome of a bacterium can also be detected by well-known methods, including PCR, Southern blotting and the like. In addition, the level of gene expression can be estimated by measuring the amount of the RNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, and the like.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

L-amino acid-producing bacteria

As a bacterium of the present invention which is modified to attenuate expression of the cynT, cynS, cynX or cynR gene, bacteria which are able to produce either an aromatic or a non-aromatic L-amino acids may be used.
The bacterium of the present invention can be obtained by attenuating expression of the cynT, cynS, cynX or cynR gene in a bacterium which inherently has the ability to produce L-amino acids. Alternatively, the bacterium of present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium already having attenuated expression of the cynT, cynS, cynX or cynR gene.

L-threonine-producing bacteria

Examples of parent strains which can be used to derive the L-threonine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) ( U.S. Patent No. 5, 175, 107 , U.S. Patent No. 5,705,371 ), E. coli 472T23/pYN7 (ATCC 98081) ( U.S. Patent No.5,631,157 ), E. coli NRRL-21593 ( U.S. Patent No. 5,939,307 ), E. coli FERM BP-3756 ( U.S. Patent No. 5,474,918 ), E. coli FERM BP-3519 and FERM BP-3520 ( U.S. Patent No. 5,376,538 ), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 ( EP 1149911 A ), and the like.
The strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the ilvA gene in this strain has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine. The strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1^st Dorozhny proezd, 1) on April 7, 1987 under the accession number VKPM B-3996.
E. coli VKPM B-5318 ( EP 0593792B ) also may be used as a parent strain to derive L-threonine-producing bacteria of the present invention. The strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage C1 repressor and PR promoter replaces the regulatory region of the threonine operon in the plasmid pVIC40 harbored by the strain. The strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number of VKPM B-5318.
Preferably, the bacterium of the present invention is additionally modified to enhance expression of one or more of the following genes:

the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
the thrB gene which codes for homoserine kinase;
the thrC gene which codes for threonine synthase;
the rhtA gene which codes for a putative transmembrane protein;
the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and
the aspC gene which codes for aspartate aminotransferase (aspartate transaminase);

The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no. NC_000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession no. NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession no. NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K-12. All three genes functions as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon ( WO2005/049808 , WO2003/097839 ).
A mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from the well-known plasmid pVIC40 which is presented in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371 .
The rhtA gene is located at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181), and is located between the pexB and ompX genes. The DNA sequence expressing a protein encoded by the ORF1 has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, it is known that the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17^th International Congress of Biochemistry and Molecular Biology in conjugation with the Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A ).
The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession no. NC_000913.1, gi: 16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.
Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession no. NC_000913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.

L-lysine-producing bacteria

Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to an L-lysine analogue. The L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine is present in the medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam and so forth. Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170 ) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.
The strain WC196 may be used as an L-lysine producing bacterium of Escherichia coli. This bacterial strain was bred by conferring AEC resistance to the strain W3110, which was derived from Escherichia coli K-12. The resulting strain was designated Escherichia coli AJ13069 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December 6, 1994 and received an accession number of FERM P-14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and received an accession number of FERM BP-5252 ( U.S. Patent No. 5,827,698 ).
Examples of parent strains which can be used to derive L-lysine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) ( U.S. Patent No. 6,040,160 ), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), and aspartase (aspA) ( EP 1253195 A ). In addition, the parent strains may have an increased level of expression of the gene involved in energy efficiency (cyo) ( EP 1170376 A ), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) ( U.S. Patent No. 5,830,716 ), the ybjE gene ( WO2005/073390 ), or combinations thereof.
Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine. Examples of the enzymes that catalyze a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase ( U.S. Patent No. 5,827,698 ), and the malic enzyme ( WO2005/010175 ).

L-cysteine-producing bacteria

Examples of parent strains which can be used to derive L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases ( U.S. Patent No. 6,218,168 , Russian patent application 2003121601 ); E. coli W3110 which over-expresses genes which encode proteins suitable for secreting substances toxic for cells ( U.S. Patent No. 5,972,663 ); E. coli strains having lowered cysteine desulfohydrase activity ( JP11155571A2 ); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene ( WO0127307A1 ), and the like.

L-leucine-producing bacteria

Examples of parent strains which can be used to derive L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121 )) or leucine analogs including β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine ( JP 62-34397 B and JP 8-70879 A ); E. coli strains obtained by the genetic engineering methods such as those described in WO96/06926 ; E. coli H-9068 ( JP 8-70879 A ), and the like.
The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which are preferably represented by a mutant leuA gene coding for isopropylmalate synthase which is not subject to feedback inhibition by L-leucine ( US Patent 6,403,342 ). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) ( EP 1239041 A2 ).

L-histidine-producing bacteria

Examples of parent strains which can be used to derive L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116 - B12121 ( U.S. Patent No. 4,388,405 ); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) ( U.S. Patent No. 6,344,347 ); E. coli H-9341 (FERM BP-6674) ( EP1085087 ); E. coli AI80/pFM201 ( U.S. Patent No. 6,258,554 ), and the like.
Examples of parent strains which can be used to derive L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth.
It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation into any of these genes which imparts resistance to the feedback inhibition to enzymes encoded by these genes (Russian Patent Nos. 2003677 and 2119536 ).
Specific examples of strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048 which have been transformed with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme ( JP 56-005099 A ), E. coli strains transformed with rht, which encoes an amino acid-exporter ( EP1016710A ), E. coli 80 strain imparted with sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536 ), and so forth.

L-glutamic acid-producing bacteria

Examples of parent strains which can be used to derive L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC⁺ ( EP 1172433 ). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in the thrC and ilvA genes ( U.S. Patent No. 4,278,765 ). A wild-type allele of the thrC gene was transferred using general transduction with a bacteriophage P1 which was grown on the wild-type E. coli strain K12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.
Examples of parent strains which can be used to derive the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains which are deficient in α-ketoglutarate dehydrogenase activity, or strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of such genes include genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), and so forth.
Examples of strains which have been modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989A , EP955368A , and EP952221A .
Examples of parent strains which can be used to derive the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), and glutamate decarboxylase (gadAB). Bacteria belonging to the genus Escherichia deficient in α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945 . Specifically, these strains include the following:

E. coli W3110sucA::Km^R
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)
E. coli W3110sucA::Km^R is obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in α-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM. BP-5807) ( U.S. Patent No. 5,908,768 ), FERM P-12379, which additionally has a low L-glutamic acid decomposing ability ( U.S. Patent No. 5,393,671 ); AJ13138 (FERM BP-5565) ( U.S. Patent No. 6,110,714 ), and the like.
Examples of L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea which are deficient in α-ketoglutarate dehydrogenase activity or have decreased α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJ13356. ( U.S. Patent No. 6,331,419 ). Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P-16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficient in α-ketoglutarate dehydrogenase activity as a result of disruption of the α-KGDH-E1 subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as Enterobacter agglomerans AJ13356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.

L-phenylalanine-producing bacteria

Examples of parent strains which can be used to derive L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197), E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene ( U.S. Patent No. 5,354,672 ), E. coli MWEC101-b (KR8903681), E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 ( U.S. Patent No. 4,407,952 ). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ12604 (FERM BP-3579) may be used ( EP 488424 B1 ). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used ( U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1 ).

L-tryptophan-producing bacteria

Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) which is deficient in tryptophanyl-tRNA synthetase encoded by mutant trpS gene ( U.S. Patent No. 5,756,345 ); E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase which is not subject to feedback inhibition by serine and a trpE allele encoding anthranilate synthase which is not subject to feedback inhibition by tryptophan ( U.S. Patent No. 6,180,373 ), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase ( U.S. Patent No. 4,371,614 ), E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced ( WO9708333 , U.S. Patent No. 6,319,696 ), and the like. L-tryptophan-producing bacteria belonging to the genus Escherichia which have enhanced activity of the protein encoded by the yedA or the yddG genes may also be used ( U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1 ).
Examples of parent strains forwhich can be used to derive the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities are enhanced of the following enzymes: anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and tryptophan synthase (trpAB). The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, therefore a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 ( WO 94/08031 ), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria of the present invention also include strains which have been transformed with the tryptophan operon containing a gene encoding desensitized anthranilate synthase ( JP 57-71397 A , JP 62-244382 A , U.S. Patent No. 4,371,614 ). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). Tryptophan synthase consists of α and β subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon ( WO2005/103275 ).

L-proline-producing bacteria

Examples of parent strains which can be used to deriving L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the ilvA gene and is able to produce L-proline ( EP 1172433 ). The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes include the proB gene coding for glutamate kinase which is desensitized to feedback inhibition by L-proline ( DE Patent 3127361 ). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins responsible for secreting L-amino acids from the bacterial cell. Such genes are exemplified by b2682 and b2683 genes (ygaZH genes) ( EP1239041 A2 ).
Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 ( GB Patent 2075056 ), VKPM B-8012 (Russian patent application 2000124295 ), plasmid mutants described in DE Patent 3127361 , plasmid mutants described by Bloom F.R. et al (The 15th Miami winter symposium, 1983, p.34), and the like.

L-arginine-producing bacteria

Examples of parent strains which can be used to derive L-arginine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925) ( U.S. Patent Application 2002/058315 A1 ) and derivative strains thereof harboring mutant N-acetylglutamate synthase (Russian Patent Application No. 2001112869 ), E. coli strain 382 (VKPM B-7926) ( EP1170358A1 ), an arginine-producing strain transformed with the argA gene encoding N-acetylglutamate synthetase ( EP1170361A1 ), and the like.
Examples of parent strains which can be used to derive L-arginine producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), carbamoyl phosphate synthetase (carAB), and so forth.

L-valine-producing bacteria

Example of parent strains which can be used to derive L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon ( U.S. Patent No. 5,998,178 ). It is desirable to remove the region of the ilvGMEDA operon responsible for attenuation so that the produced L-valine cannot attenuate expression of the operon. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.
Examples of parent strains which can be used to derive L-valine-producing bacteria of the present invention also include mutants of amino-acyl t-RNA synthetase ( U.S. Patent No. 5,658,766 ). For example, E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used. E. coli VL1970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession number VKPM B-4411.
Furthermore, mutants requiring lipoic acid for growth and/or lacking H⁺-ATPase can also be used as parent strains ( WO96/06926 ).

L-isoleucine-producing bacteria

Examples of parent strains which can be used to derive L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6-dimethylaminopurine ( JP 5-304969 A ), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate ( JP 5-130882 A ). In addition, recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase, can also be used as parent strains ( JP 2-458 A , FR 0356739 , and U.S. Patent No. 5,998,178 ).

2. Method of the present invention

The method of the present invention is a method for producing an L-amino acid by cultivating the bacterium of the present invention in a culture medium to produce and secrete the L-amino acid into the medium, and collecting the L-amino acid from the medium.
In the present invention, the cultivation, collection, and purification of an L-amino acid from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.
The chosen culture medium may be either a synthetic or natural medium, so long as it includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen microorganism, alcohol, including ethanol and glycerol, may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.
The cultivation is preferably performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40 °C, preferably 30 to 38 °C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.

Brief Description of Drawings

Figure 1 shows the relative positions of primers P1 and P2 on plasmid pACYC184, which is used for amplification of the cat gene.
Figure 2 shows the construction of the chromosomal DNA fragment comprising the inactivated cynT gene.

Examples

The present invention will be more concretely explained below with reference to the following non-limiting Examples.

Example 1. Construction of a strain with an inactivated cynT gene.

1. Deletion of the cynT gene.

The cynT gene was deleted by the method initially developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called "Red-driven integration". According to this procedure, the PCR primers P1 (SEQ ID NO: 9) and P2 (SEQ ID NO: 10), which are complementary to both the region adjacent to the cynT gene and the gene conferring antibiotic resistance, respectively, in the template plasmid, were constructed. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (GenBank/EMBL accession number X06403) was used as a template in the PCR reaction. Conditions for PCR were as follows: denaturation step:3 min at 95 °C; profile for two first cycles: 1 min at 95 °C, 30 sec at 50 °C, 40 sec at 72 °C; profile for the last 25 cycles: 30 sec at 95 °C, 30 sec at 54 °C, 40 sec at 72 °C; final step: 5 min at 72 °C.
An 1152 bp PCR product (Fig. 1) was obtained and purified in agarose gel and used for electroporation of E. coli MG1655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication. The plasmid pKD46 (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6644-45) includes a 2,154 nucleotide (31088-33241) DNA fragment of phage λ (GenBank accession No. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible P_araB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655. The strain MG165S can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, U.S.A.).
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30°C in LB medium containing ampicillin (100 mg/l), and the culture was diluted 100 times with 5 ml of SOB medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30 °C to an OD₆₀₀ of ≈0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H₂O. Electroporation was performed using 70 µl of cells and ≈100 ng of PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) at 37 °C for 2.5 hours and were then plated onto L-agar containing chloramphenicol (30 µg/ml) and grown at 37 °C to select Cm^R recombinants. Then, to eliminate the pKD46 plasmid, 2 passages on L-agar with Cm at 42 °C were performed and the obtained colonies were tested for sensitivity to ampicillin.

2. Verification of the cynT gene deletion by PCR.

The mutants in which the cynT gene is deleted and are marked with the Cm resistance gene, were verified by PCR. Locus-specific primers P3 (SEQ ID NO: 11) and P4 (SEQ ID NO: 12) were used in PCR for verification. Conditions for PCR verification were as follows: denaturation step: 3 min at 94 °C; profile for the 30 cycles: 30 sec at 94 °C, 30 sec at 54 °C, 1 min at 72 °C; final step: 7 min at 72 °C. The PCR product obtained using parental cynT⁺ strain MG1655 as the template is 792 bp in length. The PCR product obtained using the mutant strain as the template is 1214 nucleotides in length (Fig.2). The mutant strain was named MG1655 Δ cynT::cat.

Example 2. Production of L-threonine by E. coli strain B-3996-ΔcynT

To test the effect of inactivation of the cynT gene on threonine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔcynT::cat were transferred to the threonine-producing E. coli strain VKPM B-3996 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain B-3996ΔcynT. The strain B-3996 was deposited on November 19, 1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street, 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) under the accession number VKPM B-3996.
Both E. coli strains, B-3996 and B-3996-ΔcynT, were grown for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20x200-mm test tubes. Cells were grown for 65 hours at 32°C with shaking at 250 rpm.
After cultivation, the amount of L-threonine which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol - acetic acid - water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted with 0.5 % water solution of CdCl₂, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of eight independent test tube fermentations are shown in Table 1. As follows from Table 1, B-3996-ΔcynT produced a higher amount of L-threonine, as compared with B-3996.
The composition of the fermentation medium (g/l) was as follows:

Glucose 80.0

(NH₄)₂SO₄ 22.0

NaCl 0.8

KH₂PO₄ 2.0

MgSO₄·7H₂O 0.8

FeSO₄·7H₂O 0.02

MnSO₄·5H₂O 0.02

Thiamine HCl 0.0002

Yeast extract 1.0

CaCO₃ 30.0
Glucose and magnesium sulfate were sterilized separately. CaCO₃ was sterilized by dry-heat at 180°C for 2 hours. The pH was adjusted to 7.0. Table 1

Strain OD₅₄₀ Amount of L-threonine, g/l

B-3996 23.7 ± 0.8 25.8 ± 0.3

B-3996-ΔcynT 21.8 + 1.2 26.7 ± 0.6
Hereinafter, the geneX mentioned in the Examples described below means cynT gene, cynS gene, cynX gene, cynR gene, or combinations of thereof, including cynTSX operon, cynTSX operon plus cynR gene.

Example 3. Construction of a strain with an inactivated geneX.

1. Deletion of the geneX.

The geneX can be deleted by the method initially developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called "Red-driven integration". According to this procedure, the PCR primers, which are complementary to both the region adjacent to the geneX and the gene conferring antibiotic resistance in the template plasmid, respectively, can be constructed by the analogy to the Example 1 (Figure 1). The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (GenBank/EMBL accession number X06403) can be used as a template in the PCR reaction. Conditions for PCR can be as described in the Example 1.
A PCR product can be obtained and purified in agarose gel and used for electroporation of E. coli MG1655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication. The plasmid pKD46 (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide (31088-33241) DNA fragment of phage λ (GenBank accession No. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible P_araB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655.
Electrocompetent cells can be prepared and electroporation can be performed as described in the Example 1. Cells after electroporation can be incubated with 1 ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) at 37 °C for 2.5 hours and can be then plated onto L-agar containing chloramphenicol (30 µg/ml) and grown at 37 °C to select Cm^R recombinants. Then, to eliminate the pKD46 plasmid, 2 passages on L-agar with Cm at 42 °C can be performed and the obtained colonies can be tested for sensitivity to ampicillin.

2. Verification of the geneX deletion by PCR.

The mutants in which the geneX is deleted and are marked with the Cm resistance gene, can be verified by PCR. Locus-specific primers can be used in PCR for verification. Conditions for PCR verification can be as described in the Example 1. The PCR product obtained using the parental X⁺ strain MG1655 as the template, must be longer in length then the PCR product obtained using the mutant strain as the template. The mutant strain can be named MG1655 ΔgeneX::cat.

Example 4. Production of L-threonine by E. coli strain B-3996-ΔgeneX

To test the effect of inactivation of the geneX on threonine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔgeneX::cat can be transferred to the threonine-producing E. coli strain VKPM B-3996 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain B-3996ΔX.
Both E. coli strains, B-3996 and B-3996-ΔgeneX, can be grown as described in the Example 2.
After cultivation, the amount of L-threonine, which had accumulated in the medium, can be determined by paper as described in the Example 2.

Example 5. Production of L-lysine by E. coli strain AJ11442-ΔgeneX

To test the effect of inactivation of the geneX on lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔX::cat can be transferred to the lysine-producing E. coli strain AJ11442 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain AJ11442-ΔgeneX. The strain AJ14442 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and received an accession number of FERM P-5084. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on October 29, 1987, and received an accession number of FERM BP-1543.
Both E. coli strains, AJ11442 and AJ11442-ΔgeneX, can be cultured in L-medium at 37°C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500-ml flask. The cultivation can be carried out at 37°C for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm.
After the cultivation, the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.
The composition of the fermentation medium (g/l) is as follows:

Glucose 40

(NH₄)₂SO₄ 24

K₂HPO₄ 1.0

MgSO₄·7H₂O 1.0

FeSO₄·7H₂O 0.01

MnSO₄·5H₂O 0.01

Yeast extract 2.0
The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Glucose and MgSO₄·7H₂O are sterilized separately. CaCO₃ is dry-heat sterilized at 180°C for 2 hours and added to the medium for a final concentration of 30 g/l.

Example 6. Production of L-cysteine by E. coli strain JM15(ydeD)-ΔgeneX

To test the effect of inactivation of the gene X on L-cysteine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔgeneX::cat can be transferred to the E. coli L-cysteine-producing strain JM15(ydeD) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain JM15(ydeD)-ΔgeneX.
E. coli strain JM15(ydeD) is a derivative of E. coli strain JM15 ( US Patent No. 6,218,168 ) which can be transformed with DNA having the ydeD gene, which codes for a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid ( U.S. Patent No. 5,972,663 ). The strain JM15 (CGSC# 5042) can be obtained from The Coli Genetic Stock Collection at the E.coli Genetic Resource Center, MCD Biology Department, Yale University (http://cgsc.biology.yale.edu/).
Fermentation conditions for evaluation of L-cysteine production were described in detail in Example 6 of US Patent No. 6,218,168 .

Example 7. Production of L-leucine by E. coli 57-ΔgeneX

To test the effect of inactivation of the gene X on L-leucine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔgeneX::cat can be transferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386, US Patent No. 6,124,121 ) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain 57-pMW-ΔX. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386.
Both E. coli strains, 57 and 57-ΔgeneX can be cultured for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% sucrose. Then, the fermentation medium can be inoculated with 0.21 ml of seed material (10%). The fermentation can be performed in 2 ml of a minimal fermentation medium in 20x200-mm test tubes. Cells can be grown for 48-72 hours at 32°C with shaking at 250 rpm. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol - acetic acid - water = 4:1:1).
The composition of the fermentation medium (g/l) (pH 7.2) is as follows:

Glucose 60.0

(NH₄)₂SO₄ 25.0

K₂HPO₄ 2.0

MgSO₄·7H₂O 1.0

Thiamine 0.01

CaCO₃ 25.0

Glucose and CaCO₃ are sterilized separately.

Example 8. Production of L-histidine by E. coli strain 80-ΔgeneX

To test the effect of inactivation of the gene X on L-histidine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔgeneX::cat can be transferred to the histidine-producing E. coli strain 80 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 80-ΔgeneX. The strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on October 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.
Both E. coli strains, 80 and 80-ΔgeneX, can each be cultured in L-broth for 6 h at 29°C. Then, 0.1 ml of obtained culture can be inoculated into 2 ml of fermentation medium in a 20x200-mm test tube and cultivated for 65 hours at 29°C with shaking on a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase consisting of n-butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.

The composition of the fermentation medium (g/l) is as follows (pH 6.0):

Glucose	100.0
Mameno (soybean hydrolysate)	0.2 of as total nitrogen
L-proline	1.0
(NH₄)₂SO₄	25.0
KH₂PO₄	2.0
MgSO₄·7H₂O	1.0
FeSO₄·7H₂O	0.01
MnSO₄	0.01
Thiamine	0.001
Betaine	2.0
CaCO₃	60.0

Glucose, proline, betaine and CaCO₃ are sterilized separately. The pH is adjusted to 6.0 before sterilization.

Example 9. Production of L-glutamate by E. coli strain VL334thrC ⁺ -ΔgeneX

To test the effect of inactivation of the gene X on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔgeneX::cat can be transferred to the E. coli L-glutamate-producing strain VL334thrC⁺ ( EP 1172433 ) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain VL334thrC⁺-ΔgeneX. The strain VL334thrC⁺ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on December 6, 2004 under the accession number (VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004.
Both strains, VL334thrC^- and VL334thrC⁺-ΔgeneX, can be grown for 18-24 hours at 37°C on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2ml of fermentation medium. The fermentation medium contains glucose (60g/l), ammonium sulfate (25 g/l), KH₂PO₄ (2g/l), MgSO₄ (1 g/l), thiamine (0.1 mg/ml), L-isoleucine (70 µg/ml), and CaCO₃ (25 g/l). The pH is adjusted to 7.2. Glucose and CaCO₃ are sterilized separately. Cultivation can be carried out at 30°C for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl₂.

Example 10. Production of L-phenylalanine by E. coli strain AJ12739-ΔgeneX

To test the effect of inactivation of the gene X on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔgeneX::cat can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain AJ12739-ΔgeneX. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on November 6, 2001 under accession no. VKPM B-8197 and then converted to a deposit under the Budapest Treaty on August 23,2002.
Both strains, AJ12739-ΔgeneX and AJ12739, can be cultivated at 37°C for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20x200-mm test tube and cultivated at 37°C for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. The 10x15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing no fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol : ethylacetate : 25% aqueous ammonia: water = 40 : 40 : 7 : 16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.

The composition of the fermentation medium (g/l) is as follows:

Glucose	40.0
(NH₄)₂SO₄	16.0
K₂HPO₄	0.1
MgSO₄·7H₂O	1.0
FeSO₄·7H₂O	0.01
MnSO₄·5H₂O	0.01
Thiamine HCl	0.0002
Yeast extract	2.0
Tyrosine	0.125
CaCO₃	20.0
Glucose and magnesium sulfate are sterilized separately. CaCO₃ is dry-heat sterilized at 180° for 2 hours. The pH is adjusted to 7.0.

Example 11. Production of L- tryptophan by E. coli strain SV164 (pGH5)-ΔgeneX

To test the effect of inactivation of the gene X on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔX::cat can be transferred to the tryptophan-producing E. coli strain SV164 (pGH5) by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain the strain SV164(pGH5)-ΔgeneX. The strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine. The strain SV164 (pGH5) was described in detail in US patent No. 6,180,373 or European patent 0662143 .
Both strains, SV164(pGH5)-ΔgeneX and SV164(pGH5), can each be cultivated with shaking at 32°C for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (10 mg/l, marker of pGH5 plasmid). The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (10 mg/l) in 20 x 200-mm test tubes, and cultivated at 32°C for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 10.

The fermentation medium components are listed in Table 2, but should be sterilized in separate groups (A, B, C, D, E, F, and G), as shown, to avoid adverse interactions during sterilization.

Table 2

Solutions	Component	Final concentration, g/l
A	KH₂PO₄	0.28
	NaCl	0.14
	(NH₄)₂SO₄	16
	L-Methionine	0.08
	L-Phenylalanine	0.28
	L-Tyrosine	0.28
	Mameno (total N)	0.07
B	Glucose	40.0
B	MgSO₄·7H₂O	0.03
C	FeSO₄·7H₂O	0.03
D	Na₂MoO₄·2H₂O	0.00015
	H₃BO₃	0.0025
	CoCl₂·6H₂O	0.00007
	CuSO₄·5H₂O	0.00025
	MnCl₂·4H₂O	0.0016
	ZnSO₄·7H₂O	0.0003
E	Thiamine HCl	0.001
F	CaCO₃	30.0
G	Pyridoxine	0.03

The pH of solution A is adjusted to 7.1 with NH₄OH.

Example 12. Production of L-proline by E. coli strain 702ilvA-ΔgeneX

To test the effect of inactivation of the gene X on L-proline production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔgeneX::cat can be transferred to the proline-producing E. coli strain 702ilvA by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 702ilvA-ΔgeneX. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on July 18, 2000 under accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
Both E. coli strains, 702ilvA and 702ilvA-ΔgeneX, can be grown for 18-24 hours at 37°C on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 9.

Example 13. Production of L-arginine by E. coli strain 382-ΔgeneX

To test the effect of inactivation of the gene X on L-arginine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔX::cat can be transferred to the arginine-producing E. coli strain 382 by P1 transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY) to obtain strain 382-ΔgeneX. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on April 10, 2000 under accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
Both strains, 382-ΔgeneX and 382, can be cultivated with shaking at 37°C for 18 hours in 3 ml of nutrient broth, and 0.3 ml of the obtained cultures can be inoculated into 2 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32°C for 48 hours on a rotary shaker.
After the cultivation, the amount of L-arginine which had accumulates in the medium can be determined by paper chromatography using the following mobile phase: butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent. A spot containing L-arginine can be cut out, L-arginine can be eluted with 0.5% water solution of CdCl₂, and the amount of L-arginine can be estimated spectrophotometrically at 540 nm.
The composition of the fermentation medium (g/l) is as follows:

Glucose 48.0

(NH4)₂SO₄ 35.0

KH₂PO₄ 2.0

MgSO₄·7H₂O 1.0

Thiamine HCl 0.0002

Yeast extract 1.0

L-isoleucine 0.1

CaCO₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO₃ is dry-heat sterilized at 180°C for 2 hours. The pH is adjusted to 7.0.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.

Industrial Applicability

According to the present invention, production of an L-amino acid by a bacterium of the Enterobacteriaceae family can be enhanced.

PREFERRED EMBODIMENTS

1. An L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein said bacterium has been modified to attenuate expression of one or more of the genes selected from the group consisting of cynT, cynS, cynX and cynR.
2. The bacterium according to item 1, wherein said expression of one or more genes selected from the group consisting of cynT, cynS, cynX and cynR is attenuated by inactivation of the one more genes.
3. The bacterium according to item 1, wherein said bacterium belongs to the genus Escherichia.
4. The bacterium according to item1, wherein said bacterium belongs to the genus Pantoea.
5. The L-amino acid-producing bacterium according to any of items 1 to 4, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
6. The L-amino acid-producing bacterium according to item 5, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
7. The L-amino acid-producing bacterium according to item 5, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.
8. A method for producing an L-amino acid comprising:
- cultivating the bacterium according to any of items 1 to 7 in a medium, and
- collecting said L-amino acid from the medium.
9. The method according to item 8, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
10. The method according to item 9, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
11. The method according to item 9, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.

Claims

An L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein said bacterium has been modified to attenuate expression of one or more of the genes selected from the group consisting of cynT, cynS, cynX and cynR,
wherein said L-amino acid is an aromatic L-amino acid.
The bacterium according to claim 1, wherein said expression of one or more genes selected from the group consisting of cynT, cynS, cynX and cynR is attenuated by inactivation of the one more genes.
The bacterium according to claim 1 or 2, wherein said bacterium belongs to the genus Escherichia.
The bacterium according to claim 1 or 2, wherein said bacterium belongs to the genus Pantoea.
The L-amino acid-producing bacterium according to any one of claims 1 to 4, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
A method for producing an L-amino acid comprising:
- cultivating the bacterium according to any of claims 1 to 5 in a medium, and

- collecting said L-amino acid from the medium, wherein said L-amino acid is an aromatic L-amino acid.
The method according to claim 6, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.